重组L-门冬酰胺酶的聚乙二醇化学修饰及修饰后酶的稳定性研究

目的单甲氧基聚乙二醇 (mPEG)化学修饰大肠杆菌重组L 门冬酰胺酶 (L ASP) ,考察经过修饰的酶的稳定性。方法N 羟基琥珀酰亚胺 (NHS)活化酯法活化mPEG ,生成的单甲氧基聚乙二醇琥珀酰琥珀酸亚胺酯 (SS mPEG)按不同摩尔比例与L ASP偶联 ,确定适合的反应时间和反应pH值。通过聚乙二醇化学修饰后的酶 (L ASP PEG) ,酶活力和纯度通过奈氏法和丙烯酰胺凝胶电泳 (SDS PAGE)检测 ,高效液相色谱检测L ASP PEG相对分子质量并考察了L ASP PEG体外稳定性等。结果SDS PAGE显示mPEG已经偶联到L ASP分子上 ,以两者摩尔比 1 0∶1为最佳 ,反应pH条件为 8.5 ,获得的L ASP PEG平均比活单位为 6 4 .8IU/mg ,相对分子质量为 30 1 80 0 ,体外稳定性高于L ASP。结论此实验确定了mPEG化学修饰L ASP最佳反应条件为 2 5℃反应 30min ,两者投料摩尔比为 1 0∶1 ,获得的L ASP PEG比L ASP稳定性高     

Subchronic toxicity of ethylene glycol in Wistar and F-344 rats related to metabolism and clearance of metabolites.

Ethylene glycol (CAS RN 107-21-1) can cause kidney toxicity via the formation of calcium oxalate crystals in a variety of species, including humans. Numerous repeated dose studies conducted in rats have indicated that male rats are more susceptible than female rats. Furthermore, subchronic and chronic studies using different dietary exposure regimens have indicated that male Wistar rats may be more sensitive to renal toxicity than male Fischer-344 (F-344) rats. This study was conducted to compare the toxicity of ethylene glycol in the two strains of rats under identical exposure conditions and to evaluate the potential contribution of toxicokinetic differences to strain sensitivity. Ethylene glycol was mixed in the diet at concentrations to deliver constant target dosage levels of 0, 50, 150, 500, or 1000 mg/kg/day for 16 weeks to groups of 10 male Wistar and 10 male F-344 rats based on weekly group mean body weights and feed consumption. Kidneys were examined histologically for calcium oxalate crystals and pathology. Samples of blood, urine, and kidneys from satellite animals exposed to 0, 150, 500, or 1000 mg/kg/day for 1 or 16 weeks were analyzed for ethylene glycol, glycolic acid, and oxalic acid. Treatment of Wistar rats at 1000 mg/kg/day resulted in the death of two rats; in addition, at 500 and 1000 mg/kg/day, group mean body weights were decreased compared to control throughout the 16 weeks. In F-344 rats exposed at 1000 mg/kg/day and in Wistar rats receiving 500 and 1000 mg/kg/day, there were lower urine specific gravities, higher urine volumes, and increased absolute and relative kidney weights. In both strains of rats treated at 500 and 1000 mg/kg/day, some or all treated animals had increased calcium oxalate crystals in the kidney tubules and crystal nephropathy. The effect was more severe in Wistar rats than in F-344 rats. Accumulation of oxalic acid in the kidneys of both strains of rats was consistent with the dose-dependent and strain-dependent toxicity. As the nephrotoxicity progressed over the 16 weeks, the clearance of ethylene glycol and its metabolites decreased, exacerbating the toxicity. Benchmark dose analysis indicated a BMDL05 for kidney toxicity in Wistar rats of 71.5 mg/kg/day; nearly fourfold lower than in F-344 rats (285 mg/kg/day). This study confirms that the Wistar rat is more sensitive to ethylene glycol-induced renal toxicity than the F-344 rat and indicates that metabolism or clearance plays a role in the strain differences.     

Pilot study on efficacy of reduced cathartic bowel preparation with polyethylene glycol and bisacodyl

AIM:To evaluate the efficacy of reduced cathartic bowel preparation with 2 L polyethylene glycol(PEG)-4000 electrolyte solution and 10 mg bisacodyl enteric-coated tablets for computed tomographic colonography(CTC).METHODS:Sixty subjects who gave informed consent were randomly assigned to study group A,study group B or the control group.On the day prior to CTC,subjects in study group A were given 20 mL 40% wt/vol barium sulfate suspension before 3 mealtimes,60 mL 60% diatrizoate meglumine diluted in 250 mL water after supper,and 10 mg bisacodyl enteric-coated tablets 1 h before oral administration of 2 L PEG-4000 electrolyte solution.Subjects in study group B were treated identically to those in study group A,with the exception of bisacodyl which was given 1 h after oral PEG-4000.Subjects in the control group were managed using the same strategy as the subjects in study group A,but without administration of bisacodyl.Residual stool and fluid scores,the attenuation value of residual fluid,and discomfort during bowel preparation in the three groups were analyzed statistically.RESULTS:The mean scores for residual stool and fluid in study group A were lower than those in study group B,but the differences were not statistically significant.Subjects in study group A showed greater stool and fluid cleansing ability than the subjects in study group B.The mean scores for residual stool and fluid in study groups A and B were lower than those in the control group,and were significantly different.There was no significant difference in the mean attenuation value of residual fluid between study group A,study group B and the control group.The total discomfort index during bowel preparation was 46,45 and 45 in the three groups,respectively,with no significant difference.CONCLUSION:Administration of 10 mg bisacodyl enteric-coated tablets prior to or after oral administration of 2 L PEG-4000 electrolyte solution enhances stool and fluid cleansing ability,and has no impact on the attenuation value of residual fluid or the discomfor     

Coupling of Metal Containing Homing Devices to Liposomes via a Maleimide Linker: Use of TCEP to Stabilize Thiol-groups without Scavenging Metals

Liposomes for drug delivery are often prepared with maleimide groups on the distal end of PEG to enable coupling of homing devices, such as antibodies, or other proteins. EDTA is used to stabilize the thiol group in the homing device for attachment to the maleimide. However, when using a homing device that contains a metal, EDTA inactivates this by scavenging of the metal. Holo-transferrin (Tf) containing two iron atoms (Fe³⁺), has a much higher affinity for the Tf receptor than apo-Tf (which does not contain any Fe³⁺). To couple Tf to a liposome, the introduction of a thiol group is necessary. During this process, by using N-succinimidyl S-acetylthioacetate (SATA), followed by 2–3 h coupling to the liposomes, Fe³⁺ is scavenged by EDTA. This causes a decreased affinity of Tf for its receptor, resulting in a decreased targeting efficiency of the liposomes.

Tris(2-carboxyethyl)phosphine (TCEP) hydrochloride is a sulfhydryl reductant that is often used in protein biochemistry. We found that TCEP (0.01 mM) does not scavenge Fe³⁺ from Tf and is able to protect thiol groups for the coupling to maleimide. Furthermore, TCEP does not interfere with the maleimide coupling itself.

In this communication, we describe the preparation of liposomes, focussing on the coupling of Tf to the maleimide linker at the distal end of PEG, without loosing Fe³⁺ from Tf. This method can be applied to other metal-containing homing devices as well.     

Outcomes with bilateral cochlear implantation following sudden dual sensory loss after ethylene glycol poisoning

Abstract

Acute loss of vision accompanied by profound loss of hearing is fortunately rare, but has a catastrophic effect on both the patient and their family. Re-establishing communication and spatial awareness are high priorities. We describe the case of a 45 year-old man who presented as a result of poisoning by ethylene glycol. Following assessment by clinicians who learned the deaf-blind alphabet in order to communicate, he had his hearing successfully rehabilitated with simultaneous bilateral cochlear implants. The patient recovered the ability to understand speech near perfectly in quiet, to attend to the ear giving the clearer signal in noise, and to localise sources of sound. The patient reported that the latter skill facilitated mobility. This is the first reported case of a patient with acute dual sensory loss due to ethylene glycol poisoning benefiting from bilateral cochlear implants.     

Amelioration of Endotoxin-Induced Uveitis in Rabbit by Topical Administration of Tacrolimus Proglycosome Nano-Vesicles

This work was aimed to improve the efficacy of tacrolimus in the treatment of endotoxin-induced uveitis (EIU) using propylene glycol modified lipid vesicles termed as proglycosome nano-vesicles (PNVs). PNVs were prepared by modified film hydration method. Experimental uveitis in rabbit eye was induced by an intravitreal injection of 20 μL of the endotoxin solution containing 100 ng of lipopolysaccharide endotoxin. In vivo efficacy of PNVs was determined by studying clinical symptoms of uveitis using slit lamp examination and by quantitatively measuring levels of tumor necrosis factor-alpha, interleukin-6, leukocytes and total proteins in aqueous humor, 24 h after intravitreal injection of endotoxin. Comparison was made with healthy, untreated and tacrolimus solution treated eyes. PNVs developed were nano-sized, deformable and showed sustained release of tacrolimus over period of 12 h. In vivo results indicated statistically significant difference between the effects of PNVs in the treatment of EIU compared to tacrolimus. PNV treatment not only subsides clinical symptoms of uveitis but also prevented breakdown of blood aqueous barrier. Tacrolimus loaded PNVs are potential new topical treatment for uveitis.     

不同消毒灭菌方案对纤维支气管镜活检钳的消毒效果分析

目的：探讨不同灭菌方案对纤维支气管镜活检钳的消毒效果。方法：对环氧乙烷消毒、戊二醛浸泡消毒及高压蒸汽灭菌3种消毒灭菌方案对纤维支气管镜活检钳的灭菌合格率、污染率和使用年限进行效果比较。结果：环氧乙烷消毒法的合格率高于戊二醛浸泡消毒和高压蒸汽灭菌消毒法,且污染率也显著低于另外两组,器械使用寿命显著延长。结论：低温环氧乙烷灭菌法对纤维支气管镜活检钳的灭菌效果好,且污染率较低,使用寿命更长。     

Effect of microfluidization parameters on the physical properties of PEG-PLGA nanoparticles prepared using high pressure microfluidization

The objective of this work was to develop uniformly distributed poly(ethylene glycol) grafted poly(lactide-co-glycolide) (PEG-PLGA) nanoparticles of mean size range ～100–200 nm using ethyl acetate as the solvent. In the multiple emulsion solvent evaporation method a high pressure microfluidization process was adopted to produce the W/O/W multiple emulsion. Non-toxic ethyl acetate was used to solubilize PEG-PLGA. The mean size of nanoparticles obtained was less than 180 nm. The particle size and size distribution were dependent on the microfluidization conditions applied. Mean particle size steadily increased from 121 nm at three passes to 172 nm at 20 passes of the microfluidizer, indicating that over-processing may be detrimental to PEG-PLGA nanoparticles prepared using this technique. There was no significant alteration of the PEG-PLGA matrix, as evidenced from the differential scanning calorimetric studies.     

Flexible Phase Change Material Fiber: A Simple Route to Thermal Energy Control Textiles

A flexible hollow polypropylene (PP) fiber was filled with the phase change material (PCM) polyethylene glycol 1000 (PEG1000), using a micro-fluidic filling technology. The fiber’s latent heat storage and release, thermal reversibility, mechanical properties, and phase change behavior as a function of fiber drawing, were characterized. Differential scanning calorimetry (DSC) results showed that both enthalpies of melting and solidification of the PCM encased within the PP fiber were scarcely influenced by the constraint, compared to unconfined PEG1000. The maximum filling ratio of PEG1000 within the tubular PP filament was ~83 wt.%, and the encapsulation efficiencies and heat loss percentages were 96.7% and 7.65% for as-spun fibers and 93.7% and 1.53% for post-drawn fibers, respectively. Weak adherence of PEG on the inner surface of the PP fibers favored bubble formation and aggregating at the core–sheath interface, which led to different crystallization behavior of PEG1000 at the interface and in the PCM matrix. The thermal stability of PEG was unaffected by the PP encasing; only the decomposition temperature, corresponding to 50% weight loss of PEG1000 inside the PP fiber, was a little higher compared to that of pure PEG1000. Cycling heating and cooling tests proved the reversibility of latent heat release and storage properties, and the reliability of the PCM fiber.